Treatment of ocular conditions utilizing a histone/protein deacetylase inhibitor

ABSTRACT

A method for treating an ocular disorder in a subject comprising administering a therapeutic agent-loaded carrier to an ocular site of the subject in need thereof, wherein the therapeutic agent is a histone deactylase inhibitor.

CROSS REFERENCE TO RELATED APPLICATION

This is the U.S. National Stage of International Application No.PCT/US2018/058184, filed Oct. 30, 2018, which was published in Englishunder PCT Article 21(2), which application in turn claims the benefit ofU.S. Provisional Application No. 62/578,927, filed Oct. 30, 2017, whichis incorporated herein by reference in its entirety.

BACKGROUND

The treatments for dry eye disease are based upon the condition. Mildtreatments can include lifestyle changes, such as wearing sunglasses andless exposure to drying winds. Additional therapies to aid mild tomoderate inflammation are tear substitutes. Artificial tears do providetemporary relief for patients; however, most formulations containpreservatives such as benzalkonium chloride that can cause eyeirritation and hyperosmolarity of the tear film. Also, anti-inflammatorytreatments are used for patients with severe inflammation. Thesetreatments have shown to decrease inflammation in patients, however areonly intended for short-term use, and long-term application has beenimplicated in conditions such as glaucoma and retinopathy. Othertreatment approaches include, tear duct plugs, which reduce tearturnover. However, plugs do not address the underlying cause of theinflammatory disease.

Dry eye disease (DED) is a multifactorial ocular condition,characterized by inflammation of the ocular surface and tear filminstability, which afflicts as many as 1 in 5 individuals globally.Individuals with DED suffer symptoms including blurred vision,foreign-body and/or burning sensation, light sensitivity, and in severecases, corneal ulcerations leading to vision loss. Current treatmentspredominantly address the symptoms of DED and include artificial tears,punctual occlusion with tear plugs, and ophthalmic corticosteroids.Artificial tear substitutes may provide temporary relief for patients;however, most artificial tear formulations contain preservatives, suchas benzalkonium chloride, which sometimes can cause tear filmhyperosmolarity. This adverse effect can trigger death ofmucin-producing goblet cells, leading to further ocular irritation. Tearplugs may be used with or without artificial tears to reduce tearturnover by occluding the draining tear duct; however, these punctalplugs must be inserted by a physician, and limitations include issueswith plug retention and increased risk of ocular infections. Even withregular use of artificial tears and/or punctual plugs, many patientsremain symptomatic because these palliative treatments do not addressthe underlying cause of DED. Recently, a number of studies demonstratedan inflammatory basis for DED, which led to the application of topicalcorticosteroids to treat DED. While ophthalmic corticosteroids broadlysuppress ocular inflammation and can alleviate symptoms of DED, theeffects are transient and are prescribed for short-term use.Consequently, treatment of DED typically requires long-term use ofcorticosteroids, which is associated with severe side effects, such assteroid-induced glaucoma and retinopathy. Furthermore, despitesuppressing production of inflammatory mediators, corticosteroids do notaddress the underlying imbalance between pro-inflammatory immune cellsand immunosuppressive cells.

In DED, infiltration of pathogenic pro-inflammatory CD4⁺ T cells causesa breakdown in immunological homeostasis, ultimately compromising thelacrimal functional unit (LFU), which includes the cornea, conjunctiva,lacrimal glands, meibomian glands, and the interconnecting innervation.As T cells proliferate in the ocular tissues, these cells secretepro-inflammatory cytokines, such as IFN-γ, which inhibit naturallysuppressive immune cells known as regulatory T cells (Tregs). Thisultimately leads to a shift in the immunological balance betweentissue-protective Tregs and tissue-destructive pro-inflammatory(effector) T cells. Since the importance of Tregs contributing toimmunological tolerance has become evident over the years, investigatorshave examined methods to utilize these immunosuppressive cells. Notably,adoptive transfer of Tregs from mice with DED can suppress inflammationin a T-cell deficient nude mouse administered effector T-cells from aDED mouse. Moreover, due to the low population of Tregs found in thehuman body (5-15%), application of ex vivo transfer of Tregs has beenproposed as a method of therapeutic modulation in order to enhance thelimited numbers of Tregs. Despite such evidence suggesting thatenhancing Treg populations ex vivo is a viable therapeutic approach,there are many hurdles associated with translating a cellular therapy tothe clinic. These include expansion, contamination, and the potentialhazard of Tregs differentiating into conventional T cells.

SUMMARY

Disclosed herein is a method for treating an ocular disorder in asubject comprising administering a therapeutic agent-loaded carrier toan ocular site of the subject in need thereof, wherein the therapeuticagent is a histone deactylase inhibitor.

Also disclosed herein is a method for treating Sjögren's syndrome in asubject comprising administering a therapeutic agent-loaded carrier toan ocular site of the subject in need thereof, wherein the therapeuticagent is a histone deactylase inhibitor.

Additionally disclosed herein is a composition comprising therapeuticagent-loaded microparticles, wherein the therapeutic agent is a histonedeactylase inhibitor.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of mechanisms of action forsuberoylanilide hydroxamic acid (SAHA).

FIG. 2 is a graph showing the controlled release of SAHA.

FIG. 3 is a graph showing tear secretion.

FIG. 4 is a graph showing goblet cell density and histological sectionsof the conjunctiva.

FIG. 5 shows fluorescein staining of the corneal epithelium.

FIG. 6 are graphs showing IL-12, IFN-γ, IL-6, and FoxP3⁺ Tregs levels.

FIG. 7 are scanning electron micrographs showing the morphology ofmicroparticles and graphs showing the average size distribution ofmicroparticles.

DETAILED DESCRIPTION Terminology

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats.

The term “co-administration” or “co-administering” refers toadministration of an agent disclosed herein with at least one othertherapeutic or diagnostic agent within the same general time period, anddoes not require administration at the same exact moment in time(although co-administration is inclusive of administering at the sameexact moment in time). Thus, co-administration may be on the same day oron different days, or in the same week or in different weeks. In certainembodiments, a plurality of therapeutic and/or diagnostic agents may beco-administered by encapsulating the agents within the microparticlesdisclosed herein.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

“Microparticle”, as used herein, unless otherwise specified, generallyrefers to a particle of a relatively small size, but not necessarily inthe micron size range; the term is used in reference to particles ofsizes that can be, for example, administered to the eye in the form ofan eye drop that can be delivered from a squeeze nozzle container, andthus can be less than 50 nm to 100 microns or greater. In certainembodiments, microparticles specifically refers to particles having adiameter from about 200 nm to 30 microns, or about 1 to about 25microns, preferably from about 10 to about 25 microns, more preferablyfrom about 10 to about 20 microns. In one embodiment, the particles havea diameter from about 1 to about 10 microns, preferably from about 1 toabout 5 microns, more preferably from about 2 to about 5 microns. Asused herein, the microparticle encompasses microspheres, microcapsules,microparticles, microrods, nanorods, nanoparticles, or nanospheresunless specified otherwise. A microparticle may be of compositeconstruction and is not necessarily a pure substance; it may bespherical or any other shape.

“Ocular region” or “ocular site” means any area of the eye, includingthe anterior and posterior segment of the eye, and which generallyincludes, but is not limited to, any functional (e.g., for vision) orstructural tissues found in the eyeball, or tissues or cellular layersthat partly or completely line the interior or exterior of the eyeball.Ocular regions include the anterior chamber, the posterior chamber, thevitreous cavity, the choroid, the suprachoroidal space, the subretinalspace, the conjunctiva, the subconjunctival space, the episcleral space,the intracorneal space, the epicorneal space, the sclera, the parsplana, surgically-induced avascular regions, the macula, the retina, andthe lacrimal functional unit (LFU), which includes the cornea,conjunctiva, lacrimal glands, meibomian glands, and the interconnectinginnervation.

“Ocular condition” means a disease, ailment or condition which affectsor involves the eye or one of the parts or regions of the eye. Broadlyspeaking the eye includes the eyeball and the tissues and fluids whichconstitute the eyeball, the periocular muscles (such as the oblique andrectus muscles) and the portion of the optic nerve which is within oradjacent to the eyeball.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. Ideally, a therapeutically effective amount of an agentis an amount sufficient to inhibit or treat the disease or conditionwithout causing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition. For example, a“therapeutically effective amount” may be a level or amount of agentneeded to treat an ocular condition, or reduce or prevent ocular injuryor damage without causing significant negative or adverse side effectsto the eye or a region of the eye

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, or administering a compound or composition to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing a pathology or condition,or diminishing the severity of a pathology or condition. As used herein,the term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, an improvement in the overall healthor well-being of the subject, or by other parameters well known in theart that are specific to the particular disease. The phrase “treating adisease” refers to inhibiting the full development of a disease, forexample, in a subject who is at risk for a disease such as dry eyedisease. “Preventing” a disease or condition refers to prophylacticadministering a composition to a subject who does not exhibit signs of adisease or exhibits only early signs for the purpose of decreasing therisk of developing a pathology or condition, or diminishing the severityof a pathology or condition. In certain embodiments, “treating” meansreduction or resolution or prevention of an ocular condition, ocularinjury or damage, or to promote healing of injured or damaged oculartissue

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

The development of formulations that can induce endogenous Tregs in vivousing a single small molecule therapeutic agent would drasticallydiminish concerns of clinical safety and regulations. One particularpotential category of drugs that could serve to simplify clinicaltranslation is a class of small molecules known as histone deacetylaseinhibitors (HDACi). Specifically, this class of small molecules areknown to induce differentiation and cell cycle arrest in cancer cells.An HDACi known as suberoylanilide hydroxamic acid (SAHA)(N-hydroxy-N′-phenyl-octanediamide; vorinostate) has been approved bythe FDA for cutaneous T cell lymphoma (Commercially referred to asZolinza®; Merck & Co., Inc., Whitehouse Station, N.J.). In addition, toits usage as an anti-cancer therapeutic, this small molecule drug hasbeen attracting interest as a potential anti-inflammatory therapeutic.Moreover, HDACi have recently shown to enrich/enhance the localpopulations of effector T cells and Tregs. Specifically, the HDACi,SAHA, promotes Foxp3 acetylation, thereby increasing the binding ofFoxp3 to DNA and enhancing suppressive functions of natural Tregs(nTregs) as shown in FIG. 1. Also, SAHA can also induce the generationof tolerogenic APCs via acetylation and activation of STAT-3, which canthen lead to differentiation of induced Tregs (iTregs) as shown below inFIG. 1.

Given that a HDACi can both expand Tregs and enhance theirimmunosuppressive function, we believe that a HDACi will effectivelycause the local induction of Tregs and prevent clinical symptomsassociated with DED. Accordingly, disclosed herein are compositions thatcan sustainably release a HDACi in order to modulate Treg responseslocally for DED. Specifically, a local controlled delivery system wasutilized to promote the peripheral conversion of naïve CD4⁺ T cells intoiTregs. The compositions can encapsulate and locally release a HDACi,and thus may be able to prevent damage to the ocular tissue, enhancemRNA FoxP3 expression, and reduce the pro-inflammatory microenvironmentobserved in DED.

The controlled release formulations disclosed herein are highlyeffective at reducing the proinflammatory milieu in the ocular tissue,which is essential to the maintenance of immunological homeostasis toensure the prevention of chronic inflammation. Overall, the currenttopical therapeutics focus on acting as antagonists (ex: Xiidra) tohinder a specific cell involved in the pathogenesis of inflammatory eyediseases. While on the other hand, the use of these microparticles is anapproach to restore the homeostatic balance instead of solely targetinga pathogenic cell. Therefore, this treatment will resolve the underlyingetiology of the disease and provide a long-term ophthalmic drug deliverysystem. The methods and compositions disclosed herein also could resultin a dramatic increase of patient compliance and reduce diseasemorbidity.

In particular, disclosed herein are methods and compositions fortreating ocular disorders. In certain embodiments, the ocular disorderis a non-infectious ocular disease. Illustrative disorders include, butare not limited to, dry eye disease, uveitis, allergic conjunctivitis,scleritis and Age-Related Macular Degeneration (AMD). In certainembodiments, the disorder is an inflammatory mediated ocular disorder,particularly in cases with chronic inflammation as an underlying cause.

By ameliorating inflammation in the lacrimal glands, the therapeuticagent-loaded carrier approach disclosed herein also could be used to beused to treat Sjögren's syndrome, an autoimmune disease targeting thelacrimal and salivary glands that causes a severe form of DED and drymouth.

The methods include administering a therapeutic agent-loaded carrier toa subject, wherein the therapeutic agent includes a histone deactylaseinhibitor (HDACi). The carrier may be in the form of a thin film, a rod,contact lens, or microparticles. In certain embodiments, thecompositions include therapeutic agent-loaded microparticles.

Illustrative therapeutic agents include a subset of inhibitors that area small molecule pan-HDACi (act on all isoforms of the zinc-dependentenzymes) that may cause epigenetic modifications that regulate geneexpression and protein function in order to modulate the function ofimmune cells such as T lymphocytes. In particular, HDACi can stimulatethymic production of anti-inflammatory Tregs, enhance Treg suppressivefunction, and promote the peripheral conversion of CD4⁺ naïve T cellsinto Tregs.

Illustrative histone deactylase inhibitors include suberoylanilidehydroxamic acid (SAHA) (N-hydroxy-N′-phenyl-octanediamide); trichostatinA (TsA); entinostat (MS-275); panobinostat (LBH589); mocetinostat(MGCD); romidepsin (FK228, Depsipeptide); belinostat (PXD101); MC1568;givinostat (ITF2357); quisinostat (JNJ-26481585) 2HCl; droxinostat;AR-42; tacedinaline (CI994); valproic acid sodium salt (Sodiumvalproate); tacedinaline (CI994), Sodium butyrate; resminostat;divalproex sodium; sodium phenylbutyrate; tubastatin A; scriptaid;TMP269; BRD73954; LMK-235; (−)-parthenolide; nexturastat A; CAY10603;4SC-202; BG45; and ITSA-1 (ITSA1). In certain embodiments, the histonedeactylase inhibitor is suberoylanilide hydroxamic acid (SAHA)(N-hydroxy-N′-phenyl-octanediamide).

In certain embodiments, the therapeutic agents are highly effective atinducing and/or enhancing the immunosuppressive function of Tregs, whichis essential to the maintenance of immunological homeostasis to ensurethe prevention of chronic inflammation and autoimmunity.

In one embodiment, disclosed herein are therapeutically-relevant,modular platforms to deliver therapeutic agents in vivo by artificialparticles into the vicinity of Tregs. In one embodiment, the deliveredagents modulate Treg cell proliferation. In one embodiment, thedelivered factors modulate Treg cell immunosuppressive capacity.

In one embodiment, the method comprises introducing artificialmicroparticles in vivo wherein Tregs are recruited and/or activated. Inone embodiment, the Treg cell recruitment and/or activation inducesbiological homeostasis thus resolving the ocular disease or condition.

In certain embodiments, the amount of agent loaded into themicroparticles may range from 1 ng to 1 mg, more particularly 1 to 100μg, and most particularly, 20 to 30 μg agent per mg of microparticles.In certain specific embodiments, the amount of agent loaded into themicroparticles is 25-30 μg agent per mg of microparticles.

The polymers for the microparticle may be bioerodible polymers so longas they are biocompatible. Preferred bio-erodible polymers arepolyhydroxyacids such as polylactic acid and copolymers thereof.Illustrative polymers include poly glycolide, poly lactic acid (PLA),and poly (lactic-co-glycolic acid) (PLGA). Another class of approvedbiodegradable polymers is the polyhydroxyalkanoates.

Other suitable polymers include, but are not limited to: polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate, cellulose propionate, cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene polyethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate), poly vinylchloride polystyrene, polyvinylpryrrolidone, alginate,poly(caprolactone), dextran and chitosan.

The percent loading of an agent may be increased by “matching” thehydrophilicity or hydrophobicity of the polymer to the agent to beencapsulated. In some cases, such as PLGA, this can be achieved byselecting the monomer ratios so that the copolymer is more hydrophilicfor hydrophilic drugs or less hydrophilic for hydrophobic drugs.Alternatively, the polymer can be made more hydrophilic, for example, byintroducing carboxyl groups onto the polymer. A combination of ahydrophilic drug and a hydrophobic drug can be encapsulated inmicroparticles prepared from a blend of a more hydrophilic PLGA and ahydrophobic polymer, such as PLA.

A preferred polymer is a PLGA copolymer or a blend of PLGA and PLA. Themolecular weight of PLGA is from about 10 kD to about 80 kD, morepreferably from about 10 kD to about 35 kD. The molecular weight rangeof PLA is from about 20 to about 30 kDa. The ratio of lactide toglycolide is from about 75:25 to about 50:50. In one embodiment, theratio is 50:50.

Illustrative polymers include, but are not limited to,poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(a)=10 kDa, acid-terminated, referred to as 502H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(a)=25 kDa, acid-terminated, referred to as 503H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(a)=30 kDa, acid-terminated, referred to as 504H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(a)=35 kDa, ester-terminated, referred to as 504); andpoly(D,L-lactic-co-glycolic acid) (PLGA, 75:25 lactic acid to glycolicacid ratio, M_(a)=10 kDa, referred to as 752).

In certain embodiments, the polymer is an ester-terminated PLGA.

In certain embodiments, the polymer is a polyethyleneglycol-poly(lactic-co-glycolic acid) copolymer.

In certain embodiments, the polymer microparticles are biodegradable.

In certain embodiments, the agent-loaded microparticles may have avolume average diameter of 200 nm to 30 μm, more particularly 1 to 30μm. In certain embodiments, the agent-loaded microparticles do not havea volume average diameter of 10 μm or greater since such largerparticles are difficult to eject from a container in the form of an eyedrop. The agent-loaded microparticles may be pore less or they maycontain varying amounts of pores of varying sizes, typically controlledby adding NaCl during the synthesis process.

In certain embodiments, the agent-loaded microparticle-containingcomposition does not include a hydrogel, particularly a thermoresponsivehydrogel.

The agent-loaded microparticle fabrication method can be single ordouble emulsion depending on the desired encapsulated agent solubilityin water, molecular weight of polymer chains used to make themicroparticles (MW can range from ˜1000 Da to over 100,000 Da) whichcontrols the degradation rate of the microparticles and subsequent drugrelease kinetics.

The microparticle disclosed herein may provide for sustained release ofan agent. For example, the sustained release may be over a period of atleast one day, more particularly at least 5 days or at least 10 days,and most particularly at least 30 days. The agent release can be linearor non-linear (single or multiple burst release). In certainembodiments, the agent may be released without a burst effect. Forexample, the sustained release may exhibit a substantially linear rateof release of the therapeutic agent in vivo over a period of at leastone day, more particularly at least 5 days or at least 10 days, and mostparticularly at least 30 days. By substantially linear rate of releaseit is meant that the therapeutic agent is released at a rate that doesnot vary by more than about 20% over the desired period of time, moreusually by not more than about 10%. It may be desirable to provide arelatively constant rate of release of the agent from the deliverysystem over the life of the system. For example, it may be desirable forthe agent to be released in amounts from 0.1 to 100 μg per day, moreparticularly 1 to 10 μg per day, for the life of the system. However,the release rate may change to either increase or decrease depending onthe formulation of the polymer microparticle. In certain embodiments,the delivery system may release an amount of the therapeutic agent thatis effective in providing a concentration of the therapeutic agent inthe eye in a range from 1 ng/ml to 200 μg/ml, more particularly 1 to 5μg/ml. In certain embodiments, there is no initial lag phase of release.The desired release rate and target drug concentration can varydepending on the particular therapeutic agent chosen for the drugdelivery system, the ocular condition being treated, and the subject'shealth.

The microparticle disclosed herein may provide for controlled release ofan agent. The term “controlled release” as used herein, refers to theescape of any attached or encapsulated factor at a predetermined rate.For example, a controlled release of an agent may occur resulting fromthe predicable biodegradation of a polymer particle (i.e., for example,an artificial antigen presenting cell). The rate of biodegradation maybe predetermined by altering the polymer composition and/or ratioscomprising the particle. Consequently, the controlled release may beshort term or the controlled release may be long term. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year.

In certain embodiments the agent-loaded microparticles may be includedin a composition suitable for topical administration in the form of aliquid eye drop. The eye drop(s) may be administered to any ocularstructure. The eye drops may be self-administered by the subject. Theeye drop will conform comfortably to the conjunctival sac and releasethe loaded agent. The eye drop may be administered on a regimen whereinthe interval between successive eye drops is greater than at least oneday (although in certain embodiments the eye drop may be administeredonce daily or more than once daily). For example, there may be aninterval of at least 5 days, at least one week, or at least one monthbetween administrations of an eye drop(s). The agent-loadedmicroparticles disclosed herein drastically decreases the dosingfrequency (thereby increasing the likelihood of patient compliance andrecovery/prevention of worsening symptoms), it does so while avoidingclinician involvement for administration by being completelynoninvasive.

The microparticle-containing composition disclosed herein may include anexcipient component, such as effective amounts of buffering agents, andantioxidants to protect a drug (the therapeutic agent) from the effectsof ionizing radiation during sterilization. Suitable water solublebuffering agents include, without limitation, alkali and alkaline earthcarbonates, phosphates, bicarbonates, citrates, borates, acetates,succinates and the like, such as sodium phosphate, citrate, borate,acetate, bicarbonate, carbonate and the like. These agents areadvantageously present in amounts sufficient to maintain a pH of thesystem of between about 2 to about 9 and more preferably about 4 toabout 8. As such the buffering agent may be as much as about 5% byweight of the total system. Suitable water soluble preservatives includesodium bisulfate, sodium bisulfate, sodium thiosulfate, ascorbate,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuricacetate, phenylmercuric borate, phenylmercuric nitrate, parabens,methylparaben, polyvinyl alcohol, benzyl alcohol, phenylethanol and thelike and mixtures thereof. These agents may be present in amounts offrom 0.001 to about 5% by weight and preferably 0.01 to about 2% byweight.

In certain embodiments, the microparticles disclosed herein may beadministered via injection. Injection sites include but are not limitedto intraorbital lacrimal gland, extraorbital lacrimal gland,intraorbital injection, subconjunctival, intravitreal, posterior andanterior chambers of the eye.

Examples

A controlled release formulation encapsulating SAHA was fabricated toprovide sustained local delivery to the ocular tissue. The formulationwas developed using PLGA and engineered to release SAHA over the courseof one-week shown in FIG. 2. Notably, the data suggest that the localadministration of the SAHA MS in the ocular tissue was able to preventclinical signs of DED. A common clinical sign of DED is a reduction ofoverall tear production, which subsequently can lead to ocular drynessand irritation. While aqueous tear secretion in ConA (diseased) andConA+Blank MS treated control mice was significantly reduced, asexpected, SAHA MS treatment prevented ConA-induced loss of aqueous tearsecretion shown in FIG. 3. In addition to preserving aqueous tearproduction, effective therapeutics should also maintain the compositionof tears by protecting gel-forming, mucin-producing goblet cells. Thesegoblet cells are located on the apical surface of the conjunctiva andproduce important/key non-aqueous components of the tears.Interestingly, loss of goblet cells in ocular tissue in DED isreportedly due to an increase in the pro-inflammatory milieu Thus, inthis study, histological sections of the conjunctiva were examined todetermine whether SAHA MS preserved mucin-producing goblet cells afterthe administration of SAHA MS. Indeed, we observed significantly greatergoblet cell density in SAHA MS-treated mice, compared to diseased (ConA)mice, as shown in FIG. 4. Since loss of aqueous tear production and/ormucin-producing goblet cells in DED ultimately leads to damage to theocular surface, we investigated whether SAHA MS, which prevented both ofthese pathological features, also maintained integrity of the cornealepithelium. Corneal integrity was assessed with fluorescein, a dye thatstains dead epithelial cells and can diffuse into areas where cellulartight junctions are compromised. Punctate staining was observed in micewith DED induced by ConA (with or without Blank MS), as shown in FIG. 5.Notably, there was a 4-fold reduction in the average fluoresceinstaining score in the SAHA MS group as compared to the ConA alone,suggesting that the HDACi was able to prevent the damage to the oculartissue initiated by ConA.

The underlying pathogenesis of DED involves effector T cells thatinfiltrate the ocular tissue and secrete pro-inflammatory cytokines,which can directly affect the health of the ocular microenvironment.Since increases in pro-inflammatory cytokines have been found in thelacrimal gland tissues of mice with DED, we investigated whether theSAHA MS-mediated prevention of clinical signs of DED was due to areduced pro-inflammatory milieu in the ocular tissue. Specifically, mRNAexpression levels of pro-inflammatory cytokines (IL-12, IFN-γ, and IL-6)in the lacrimal gland tissue was evaluated by qRT-PCR. Expression ofIL-12 was induced by ConA injection, relative to saline controls, whichmay be attributed to ConA acting as a T-cell mitogen thus enhancing theexpression of certain genes involved in T-cell activation such as IL-12.As expression of IL-12 is known to enhance the secretion of IFN-γ by Th1cells, we also observed a significant increase in IFN-γ mRNA levels inthe ConA group as shown in FIG. 6. Both of these findings in theConA-induced murine model of DED are consistent with previous reportsindicating that IL-12 and IFN-γ are upregulated in tears of patientswith DED. In addition to increases in IL-12 and IFN-γ in the lacrimalgland, IL-6 was also significantly elevated in the ConA treatment group,which is consistent with other reports of increased IL-6 production inlacrimal glands of DED murine models. Importantly, treatment with SAHAMS significantly inhibited the ConA-induced expression of IL-12, IFN-γ,and IL-6 shown in FIG. 6. These reductions in the expression levels ofpro-inflammatory cytokines sparked the question of whether the SAHA MSwere potentially altering the immunological microenvironment byincreasing anti-inflammatory Tregs. Previous reports have demonstratedthat HDACi can expand FoxP3⁺ Tregs in vitro and in vivo. Interestingly,there was a significant rise in FoxP3 expression of Tregs locally in thelacrimal gland tissue, potentially suggesting that the reduction ofsigns of DED and pro-inflammatory microenvironment in the lacrimal glandmay be due to Tregs suppressing the ocular inflammation shown in FIG. 6.

In summary, administration of SAHA MS preserved aqueous tear productionand mucin-producing goblet cells and prevented damage to ocular tissueby reducing the pro-inflammatory milieu in the lacrimal gland andenhancing numbers and/or function of FoxP3⁺ Tregs in an inflammatorymurine model of DED.

Materials and Methods

Fabrication of HDACi Microspheres

HDACi microspheres were fabricated using a single-emulsion evaporationtechnique due to the hydrophobic nature of SAHA. Specifically, 200 mg ofPoly (lactic-co-glycolic) acid (PLGA-50:50 lactide:glycolide, acidterminated) (MW:7,000-17,000) (viscosity: 0.16-0.24 dL/g, 0.1% (w/v)(Sigma Aldrich, MO) was used to encapsulate 40 mg of SAHA (Selleck Chem,TX) in 4 ml of dichloromethane and sonicated for 1 hour. Subsequently,this emulsion was then mixed with 60 ml of 2% polyvinyl-alcohol (PVA, MW˜25,000, 98% hydrolyzed; PolySciences) and homogenized (L4RT-A,Silverson, procured through Fisher Scientific) at 3,500 rpm for 1 min.Then the homogenized mixtures were added to 80 ml of 1% PVA on a stirplate and left for approximately 1.5 hours in order for the organicsolvent to evaporate. After 1.5 hours, the microspheres were centrifuged(200 g, 5 min, 4° C.), washed 4 times with deionized water, andlyophilized for 48 hours (Virtis Benchtop K freeze dryer, Gardiner,N.Y.).

Characterization of HDACi Microspheres

The morphology of the microspheres was characterized using scanningelectron microscopy (SEM) (JEOL, JSM-6330F, Peabody, Mass.) and volumeimpedance measurements were performed on a Beckman Coulter Counter(Multisizer-3, Beckman Coulter, Fullerton, Calif.). In order todetermine the release kinetics of the SAHA microspheres, 10 mg of thefabricated microspheres with drug (SAHA) or unloaded microspheres(composed of no drug only polymer—as a control) were added to 1 ml of0.2% Tween 80 in PBS (Sigma Aldrich, St. Louis, Mo.), which was placedonto a rotator at 37° C. The supernant was sampled daily and the releaseprofile was determined using a NanoDrop 2000 Spectrophotometer(ThermoFisher Scientific, MA).

Mice

Female Balb/c mice aged 6-8 weeks were utilized for this experimentalstudy. (Charles Rivers Laboratories, Wilmington, Mass.). All murineexperiments were approved by the Institutional Animal Care and UseCommittee, University of Pittsburgh, Pittsburgh, Pa.

Murine Model

10 mg/ml of Concanavalin A (ConA) (Sigma Aldrich, St. Louis, Mo.) inphosphate buffered saline solution (PBS) was injected into the lacrimalglands with a dissecting microscope (Olympus SZX10, Waltham, Mass.) inorder to induce an inflammatory-murine model of DED. (10 mg/ml of eitherBlank MS or SAHA MS were administered together with ConA).

Tear Production

In order to measure tear production, phenol red cotton threads wereutilized. (Oasis Medical, San Dimas, Calif.). Specifically, the phenolred thread was placed in the lateral canthus of the eye for a period of60 seconds, and the amount of tears absorbed onto the thread (when tearsare absorbed onto the thread a color change occurs from yellow to red)was measured using a dissecting microscope (Olympus SZX10, Waltham,Mass.).

Corneal Fluorescein Staining

Approximately 1 μl of fluorescein (1% solution in PBS) was applied tothe conjunctival sac. Subsequently, the ocular surface was examinedusing a dissecting microscope (Olympus SZX10, Waltham, Mass.) toidentify punctate staining. The scoring of staining was completed by amasked ophthalmologist, and scored 0 for no staining, score 1 for aquarter of staining, score of 2 for less than a half, score of 3 forhalf, and 4 for more than half of the eye.

Ocular Histology

At the end of the one-week study, murine eyes were exenterated and fixedin 10% neutral buffered formalin solution for a period of 48 hours. Thenparaffin embedded sections were cut at approximately 5 μm and stainedwith Periodic Acid Schiff (PAS). These histological sections werescanned and the goblet cell density was quantified using a Zeiss AxioScan. Z1 (Thornwood, N.Y.) and Panoramic Viewer software (3D HISTECHLtd.).

qRT-PCR

Total RNA was extracted from excised lacrimal glands using TRI-reagent(Molecular Research Center, Cincinnati, Ohio), and quantified using aNanoDrop 2000 (Thermo Scientific). For the reverse transcriptase assay,2 μg RNA was converted to cDNA using a QuantiTect Reverse TranscriptionKit (Qiagen, Valencia, Calif.). Quantitative real-time PCR was thenperformed using VeriQuest Probe qPCR Mastermix (Affymetrix, Santa Clara,Calif.), (Thermo Scientific) specific for (IFN-γ:Mm01168134_m1, FAM-MGBdye), (IL-12: Mm01288989_m1, FAM-MGB dye), (IL-6:Mm00446190_m1, FAM-MGBdye), (FoxP3: Mm00475162_m1, FAM-MGB dye) and (Gusb: Mm01197698_m1,VIC-MGB PL dye, endogenous control). Duplex reactions (target gene+GUSB)were run and analyzed on a StepOnePlus Real-Time PCR System (AppliedBiosystems, Carlsbad, Calif.). Relative fold changes of IFN-γ, IL-12,IL-6, and FoxP3 expression were calculated and normalized based upon the2^(−ΔΔCt) method, with the Saline group as the untreated control.

Statistical Analysis

Data expressed as mean±S.D. Comparisons between multiple treatmentgroups were performed using one-way ANOVA, followed by Bonferronimultiple comparisons, and p≤0.05 was considered statisticallysignificant. For PCR data, a Grubb's test was performed to determine anysignificant outliers. If a significant outlier was found (P>0.05) it wasexcluded from the statistical analysis. If an assumption of the One-WayANOVA was not met a non-parametric test was performed. Statistical testswere performed using GraphPad Prism Software 6.0 (GraphPad Prism, SanDiego, Calif.).

Results

Characterization of SAHA Microspheres

SAHA MS were fabricated using the polymer (PLGA 50:50-lactic:glycolicacid, RG 502H). Scanning electron micrographs (SEM) as shown in FIG. 7illustrate that individual particles are spherical with average sizedistribution average of ˜16.956 μm (Blank Microspheres) and an averageof ˜17.099 μm (SAHA Microspheres) as confirmed by utilizing a CoulterCounter (representative plots of volume impedance measurements shownbelow in FIG. 7. Additionally, the release kinetics was characterizedusing a NanoDrop (UV-vis spectrophotometer) to detect the absorbance ofSAHA, which demonstrates a cumulative release of approximately 50 ng/mgof microsphere.

Aqueous Tear Production is Maintained with SAHA Microspheres

To determine whether SAHA MS were capable of preventing key signs ofDED, aqueous tear secretion was examined using phenol red threads at theconclusion of the experimental study.^([7]) The administration of Saline(non-diseased) did not significantly affect tear production compared toConA alone (diseased) and ConA+Blank MS as shown in FIG. 3. Markedly,the loss of tear production was prevented with the administration ofSAHA MS.

SAHA Microspheres Decrease Corneal Fluorescein Staining

A hallmark of DED is an increase of permeability to the cornealepithelial layer of the ocular tissue. Specifically, fluoresceinstaining is a standard diagnostic measurement/indicator for diseaseseverity of ocular surface damage.^([9]) Representative cornealfluorescein images were captured using a fluorescent dissectingmicroscope and scored by a masked ophthalmologist on a scale of 0 to 4,with 0 corresponding to no staining, and 4 corresponding to staining onmore than 50% of the cornea, as seen in FIG. 5. Compared to the ConAalone and ConA+Blank MS group, the uptake of fluorescein to the corneawas significantly reduced in the Saline and SAHA MS groups. Ultimately,the local administration of SAHA MS to lacrimal gland demonstrated (***p≤0.001) that the preventative therapy was able to reduce the cornealfluorescein scores by approximately %0% compared to the ConA alonegroup.

The Administration of SAHA Microspheres Prevent Loss of Goblet CellDensity

Goblet cells are located within the stratified columnar conjunctivalepithelial cells, and are known to play an integral role in producing,mucin, a component of the tears. Moreover, goblet cells have beenassociated with the production of MUC5AC, which acts as a gel layer totrap pollen and allergens. As these goblet cells are depleted due tochronic inflammation due to conditions such as DED this can ultimatelylead to conjunctival epithelial squamous metaplasia and an abnormal tearfilm. Thus, Periodic Acid Schiff (PAS staining) of the ocular tissue wasexamined to determine whether SAHA MS were able to reduce the loss ofgoblet cells compared to ConA alone thereby potentially preserving thegoblet cells (pink/purple cells) located in the conjunctiva. Notably,there was a significant preservation of goblet cell density with theadministration of SAHA MS as compared to the ConA alone group.

mRNA Expression Altered in the Lacrimal Gland with SAHA Microspheres

As inflammation occurs to the lacrimal gland, this has shown to lead toan infiltration of lymphocytes and subsequently an increase inpro-inflammatory cytokines. Thus, mRNA expression levels were examinedin the lacrimal gland tissue after ConA-induced inflammation. Data fromthe RT-PCR suggested that IL-12, IFN-γ, and IL-6 pro-inflammatorycytokines were significantly reduced in the lacrimal gland of the SAHAMS treated as compared to the ConA (diseased) group. Although,interestingly, the level of FoxP3 (transcription factor of regulatory Tcell marker) mRNA expression was significantly higher in the SAHA MSgroup as compared to the ConA (diseased) group, as seen in FIG. 6.Together, this data indicates that the SAHA MS was able to reduce thepro-inflammatory microenvironment initiated by ConA and enhance theexpression levels of FoxP3 (a transcription factor associated withTregs).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention.

What is claimed is:
 1. A composition comprising therapeutic agent-loadedmicroparticles, wherein the therapeutic agent is a histone deactylaseinhibitor, the microparticles comprise poly (lactic-co-glycolic acid),and the composition is in the form of an eye drop.
 2. The composition ofclaim 1, wherein the histone deactylase inhibitor is suberoylanilidehydroxamic acid (SAHA) (N-hydroxy-N′-phenyl-octanediamide); trichostatinA(TsA); entinostat (MS-275); panobinostat (LBH589); mocetinostat (MGCD);romidepsin (FK228, Depsipeptide); belinostat (PXD101); MC1568;givinostat (ITF2357); quisinostat (JNJ-26481585) 2HCl; droxinostat;AR-42; tacedinaline (CI994); valproic acid sodium salt (Sodiumvalproate); tacedinaline (CI994), Sodium butyrate; resminostat;divalproex sodium; sodium phenylbutyrate; tubastatin A; scriptaid;TMP269; BRD73954; LMK-235; (−)-parthenolide; nexturastat A; CAY10603;4SC-202; BG45; or ITSA-1 (ITSA1).
 3. The composition of claim 1, whereinthe histone deactylase inhibitor is suberoylanilide hydroxamic acid(SAHA).
 4. The composition of claim 1, wherein the microparticles have avolume average diameter of 1 to 30 μm.
 5. The composition of claim 1,wherein the microparticles do not have a volume average diameter of 10μm or greater.
 6. The composition of claim 1, wherein the histonedeactylase inhibitor is present in an amount of 1 ng to 1 mg, per mg ofmicroparticles.
 7. The composition of claim 1, wherein the histonedeactylase inhibitor is present in an amount of 20 to 30 μg, per mg ofmicroparticles.
 8. The composition of claim 1, wherein themicroparticles comprise a blend of poly (lactic-co-glycolic acid) andpoly lactic acid.
 9. The composition of claim 1, wherein the poly(lactic-co-glycolic acid) has a molecular weight of 10 kD to 80 kD. 10.The composition of claim 1, wherein the poly (lactic-co-glycolic acid)has a ratio of lactide to glycolide from 75:25 to 50:50.
 11. Thecomposition of claim 1, wherein the microparticles comprise anester-terminated poly (lactic-co-glycolic acid).
 12. The composition ofclaim 1, wherein the microparticles comprise a polyethyleneglycol-poly(lactic-co-glycolic acid) copolymer.
 13. The composition ofclaim 1, wherein the microparticles are biodegradable.
 14. Thecomposition of claim 1, wherein the composition has a sustained releaseof the histone deactylase inhibitor.
 15. The composition of claim 1,wherein the composition has a controlled release of the histonedeactylase inhibitor.
 16. The composition of claim 1, wherein the poly(lactic-co-glycolic acid) has a molecular weight of from about 10 kD toabout 35 kD.
 17. The composition of claim 3, wherein the poly(lactic-co-glycolic acid) has a molecular weight of from about 10 kD toabout 35 kD.